1. Field of the Invention
This invention relates to biological indicators, and in particular, to a biological indicator device and method for determining the effectiveness of sterilizing cycles.
2. Description of the Related Art
In the health care and related fields, there are many requirements for sterile equipment and devices. Similarly, the sterilization processes used in health care facilities and for bulk processing of medical devices can be applied to other sterilization processes, for example, the sterilization of chambers or large enclosures used in pharmaceutical sterility testing or in the processing of closed manufacturing lines requiring a sterile environment for filling pharmaceuticals.
Many traditional sterilization processes utilize steam or ethylene oxide (EtO), which both require pressure vessels in which the sterilization process takes place. With steam sterilization, the mode of action of steam and the effects of chamber materials, chamber design, and load configurations are well-understood, and present little impediment to achieving sterilization. During the straightforward killing of microorganisms in steam sterilization, the transfer of heat of vaporization from the steam, as it condenses on surfaces and microorganisms, causes thermal destruction of proteins essential for growth.
Similarly, EtO, which has been routinely used for many years, is well-understood. EtO, when introduced into a chamber, readily vaporizes and penetrates through most packaging and materials and kills by specifically reacting with alkyl groups on essential molecules. The ease of vaporization of EtO is due in large part to its high vapor pressure (760 mm Hg at 10.7.degree. C.). EtO is also unaffected by chamber materials such as metals and paints, and packaging materials such as synthetics and cellulosics.
Although such high-pressure, high-heat sterilization processes are still widely used, there is increased use of "cold sterilization" in which sterilization is achieved at temperatures only moderately above ambient, for example, below approximately 50.degree. C., without the need for pressure vessels as are used for steam or ethylene oxide. This process, which employs hydrogen peroxide (H.sub.2 O.sub.2), is less well understood than steam sterilization, for which there are steam tables that provide the proper time and temperature parameters required to achieve sterilizing conditions in a chamber. Although not completely understood, the mechanism of kill for H.sub.2 O.sub.2 is generally accepted as the action of hydroxyl (--OH) radicals produced when the hydrogen peroxide molecule reacts with critical organic and inorganic cell components. Other materials, such as divalent cations, catalysts, natural organic compounds, iron, brass, and the sharp edges of broken glass, can also cause the decomposition of H.sub.2 O.sub.2 and thus cause the production of --OH radicals. Since gaseous H.sub.2 O.sub.2 is reactive with many materials, each application of the technology is somewhat unique. Hydrogen peroxide is also unstable with an inherent decomposition rate in the gaseous state, which must be taken into account when using the gas for sterilization processes.
Hydrogen peroxide gas differs from the other main gaseous sterilant, EtO, in that it has a low vapor pressure (about 20 mm Hg at room temperature) under ambient conditions. Energy must be introduced to liquid H.sub.2 O.sub.2 to achieve complete and rapid vaporization and reach a sterilant concentration that can be used in a timely and efficacious manner. Once produced, the H.sub.2 O.sub.2 gas must be delivered to the site(s) to effect sterilization, in part due to the fragility of H.sub.2 O.sub.2 and in part because the H.sub.2 O.sub.2 does not readily equilibrate throughout an enclosure due to its low vapor pressure. Fans are generally used in the chamber when H.sub.2 O.sub.2 gas is used to assist in the dispersion and distribution of the sterilant.
Typically biological indicators (BIs) are used for monitoring the effectiveness of a particular sterilization process or cycle. Sterilization processes used to manufacture FDA-regulated products, such as aseptically filled sterile injectables or medical devices, must be validated for efficacy, although for some steam and EtO applications, the parametric release of specific product configurations has been established. Until a broad knowledge base exists for H.sub.2 O.sub.2, BIs will continue to be necessary to validate all applications.
Generally, a BI is a calibrated population of bacterial spores that are highly resistant to the mode of sterilization being monitored, which are positioned in or on a carrier that is placed in the sterilization chamber. Bacterial spores are both highly resistant to physical and chemical agents and are stable biological entities, so that a BI product utilizing spores has a long shelf life as compared to vegetative cells. Bacillus stearothermophilus spores have been used to monitor moist heat sterilization and gaseous hydrogen peroxide sterilization, B. subtilis spores have been used for ethylene oxide (EtO), dry heat sterilization, and liquid hydrogen peroxide processes, and B. subtilis and B. circulans have been used to monitor sterilization systems using peroxy compounds in the plasma state.
Biological indicators are typically used by placement of one or more BIs throughout a loaded enclosure, including placement in the most difficult to sterilize location(s), and then processing the load. After the sterilization process is complete, each BI is cultured as provided by the manufacturer in a bacteriological nutrient medium, and incubated at the appropriate temperature. The presence of growth in the medium after a suitable incubation period indicates that the sterilization process was not sufficient where the BI was positioned, while the absence of growth after a suitable incubation period indicates that acceptable sterilization conditions were achieved at the site of placement of the BI during the sterilization process.
The accepted validation method for determining the sterilization time for an enclosure is to place BIs inoculated at 1.times.10.sup.6 spores per carrier through the test enclosure, conduct a partial sterilization cycle, then culture and evaluate the BIs (Steris Corporation, Mentor, Ohio, Validation Manual, VHP.TM. 1000 Biodecontamination System, Part No. P-129363-317, Feb. 29, 1996). The disclosure of this manual and all other publications, including patents, that are referred to herein, is incorporated herein by reference. To check the effectiveness of a particular sterilization procedure of a particular device or chamber, this process is repeated using graduated exposure times. For exposure times shorter than are required to kill 1.times.10.sup.6 spores, outgrowth is complete from all such carriers. At some intermediate exposure times, there is a mix of positive and negative carriers (fractionals). An exposure time just long enough to kill 1.times.10.sup.6 spores on the carriers is the desired goal, since this point is recognized as the minimum level of sterilization time acceptable for many processes. Enumerating the levels of survivors on each carrier is an alternative approach, but particularly when low numbers of viable spores remain, this method is subject to some error and potential contamination through handling, and is more labor intensive than conducting multiple fractional (survival/kill) runs.
This validation procedure of sterilization processes is readily accomplished when small chambers devoid of loads, shelves and obstructions to airflow are to be sterilized, because the H.sub.2 O.sub.2 gas is easily and evenly distributed. In chambers of large size, unconventional chamber configuration, or in the presence of obstructions, however, the number of exposures needed to identify acceptable sterilization cycles may be substantially increased. Further, due to the nature of the gas and distribution difficulties, some areas of large chambers will quickly show kill, while others will require extended exposures to reach an acceptable endpoint.
Early studies using scaled sterility testing to decrease the amount of time and expense required for such testing include those reported in the patents of Kereluk (U.S. Pat. Nos. 3,711,378 and 4,087,326).
An additional problem when evaluating H.sub.2 O.sub.2 systems with various cellulosic papers such as filter paper is that H.sub.2 O.sub.2 readily adsorbs or absorbs to these materials and is difficult to remove. The retention of significant levels of H.sub.2 O.sub.2 on such materials can lead to false negative cultures due to the continued local action of H.sub.2 O.sub.2 after the gaseous sterilant source has been removed, i.e., the retained H.sub.2 O.sub.2 can continue to kill spores present on the paper carrier, thus rendering a BI that normally would have been positive (shows outgrowth) to be falsely negative (shows no outgrowth). Use of metal carriers for holding the spores overcomes these problems.
It is therefore an object of this invention to reduce the time needed to test sterilization procedures by providing a biological indicator package configured to hold multiple carriers, so that there are a plurality of incremental populations of resistant test microorganisms in each package.
It is a further object of this invention to provide a biological indicator that allows accurate, rapid assessment of sterilization processes at ambient pressures, and increased spore retention on the metal carriers. Such a carrier may be useful in sub-atmospheric applications using the gaseous hydrogen peroxide technology or other gaseous peroxy sterilization systems.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.